Von Willebrand factor (VWF) is a glycoprotein circulating in plasma as a series of multimers ranging in size from about 500 to 20,000 kD. Multimeric forms of VWF are composed of 250 kD polypeptide subunits linked together by disulfide bonds. VWF mediates initial platelet adhesion to the sub-endothelium of the damaged vessel wall. Only the larger multimers exhibit hemostatic activity. It is assumed that endothelial cells secrete large polymeric forms of VWF and those forms of VWF which have a low molecular weight (low molecular weight VWF) arise from proteolytic cleavage. The multimers having large molecular masses are stored in the Weibel-Pallade bodies of endothelial cells and liberated upon stimulation.
VWF is synthesized by endothelial cells and megakaryocytes as prepro-VWF that consists to a large extent of repeated domains. Upon cleavage of the signal peptide, pro-VWF dimerizes through disulfide linkages at its C-terminal region. The dimers serve as protomers for multimerization, which is governed by disulfide linkages between the free end termini. The assembly to multimers is followed by the proteolytic removal of the propeptide sequence (Leyte et al., Biochem. J. 274 (1991), 257-261).
The primary translation product predicted from the cloned cDNA of VWF is a 2813-residue precursor polypeptide (prepro-VWF). The prepro-VWF consists of a 22 amino acid signal peptide and a 741 amino acid propeptide, with the mature VWF comprising 2050 amino acids (Ruggeri Z. A., and Ware, J., FASEB J., 308-316 (1993)).
Defects in VWF are causal to Von Willebrand disease (VWD), which is characterized by a more or less pronounced bleeding phenotype. VWD type 3 is the most severe form in which VWF is completely missing, and VWD type 1 relates to a quantitative loss of VWF and its phenotype can be very mild. VWD type 2 relates to qualitative defects of VWF and can be as severe as VWD type 3. VWD type 2 has many sub forms, some being associated with the loss or the decrease of high molecular weight multimers. Von Willebrand syndrome type 2a (VWS-2A) is characterized by a loss of both intermediate and large multimers. VWS-2B is characterized by a loss of highest-molecular-weight multimers. Other diseases and disorders related to VWF are known in the art.
The removal or inactivation of non-lipid enveloped viruses from therapeutic protein solutions has traditionally been accomplished by treatment with physical methods like high temperature (e.g., dry heat, vapor heat, pasteurization), irradiation with high energy rays (e.g., ultraviolet (UV) rays or beta radiation), low pH, nanofiltration or by chromatographic procedures, in particular affinity chromatography. However, these procedures are often ineffective when purifying a high molecular weight protein such as VWF which does not pass through a nanofilter and/or loses its potency or molecular integrity upon treatment with heat or radiation.
Current regulatory guidelines ask manufacturers to address the issue of reduction and/or inactivation of both lipid enveloped and non-lipid enveloped viruses for recombinant pharmaceutical products. The ICH “Guideline on Viral Safety Evaluations of Biotechnology Products” (Federal Register, 1998, 63(185): 51074-51084) gives manufacturers flexibility how to address viral issues taking into account the type of product, the production process and the risk of potentially contaminating viruses. These guidelines point out that the risk of viral contamination is a feature common to all biotechnology products derived from cell lines. Such contamination could have serious clinical consequences and can arise from the contamination of the source cell lines themselves (cell substrates) or from adventitious introduction of virus during production.
Whereas the inactivation of lipid-enveloped viruses can be performed very effectively by a solvent/detergent (S/D) treatment approach, the inactivation or removal of non-lipid-enveloped model viruses (NLEV's) can be challenging due to their small size and physical stability.
Thus there exists a need in the art to develop methods to efficiently inactivate or remove non-lipid enveloped viruses during the purification of VWF.